Journal Article

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-<i>ras</i>-transformed human breast cell lines

Meng Guo and John J. Reiners

in Carcinogenesis

Volume 21, issue 7, pages 1303-1312
Published in print July 2000 | ISSN: 0143-3334
Published online July 2000 | e-ISSN: 1460-2180 | DOI:
Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-ras-transformed human breast cell lines

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The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G1 arrest that lasted ~16–24 h (IC50 ~0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3–7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor β (TGFβ) from the culture medium, supplementing the culture medium with anti-TGFβ or soluble TGFβII receptor, or pre-absorption of conditioned medium with anti-TGFβ all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by ~50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA ~50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for ~72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGFβ supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGFβII receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Keywords: ER, estrogen receptor; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; rTGFβII, transforming growth factor β type II receptor; TGFβ, transforming growth factor β; tPA, tissue plasminogen activator; TPA, 12-O-tetradecanoylphorbol-13-acetate; uPA, urokinase plasminogen activator.

Journal Article.  8328 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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